Stable diploid human fibroblasts (WI-38) will be synchronized by accumulating the cells in the G0 phase of the cell cycle. The cells will be stimulated to enter the cell cycle, and DNA will be isolated from cells in G0, G1, S and G2 phases. The DNA will be analyzed for structural modifications such as introduction of nicks and gaps, to determine how DNA is activated prior to actual replication. The DNA will also be examined for alterations in secondary structure to obtain clues as to the mechanisms of replication. The formation and subsequent metabolic fate of the various intermediates of DNA synthesis will be monitored by pulse-chase radioisotopic techniques. Each of the intermediates will be characterized as to size, secondary structure and interconversions. The long term goal is to compare mechanisms of the initiation of DNA synthesis and DNA replication, in normal and abnormal cells.